Many heterologous recombinant proteins are produced in a misfolded insoluble form, called inclusion bodies, when expressed in bacterial systems. In general, denaturing reagents must be used to solubilize the recombinant protein in the inclusion bodies. The protein must then be renatured, under conditions that have been optimized for the protein to properly fold. Efforts expended on optimization, as well as the slow refolding process and lowered process yields, add cost and time to the production of a recombinant protein.
Interferons exhibit antiviral, antiproliferative, immunomodulatory, and other activities. Several distinct types of human interferons, including α, β, and γ, have been distinguished based on, e.g., their anti-viral and anti-proliferative activities. Interferon secretion is induced by signals, including viruses, double-stranded RNAs, other polynucleotides, antigens, and mitogens. Interferon-β is an example of a protein that has been expressed in recombinant form in bacteria, where it is sequestered in inclusion bodies.
Human interferon-β 1b is a regulatory polypeptide having a molecular weight of about 22 kDa and consisting of 165 amino acid residues. It can be produced by many cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologics. It binds to a multimeric cell surface receptor. Productive receptor binding results in a cascade of intracellular events leading to the expression of interferon-β inducible genes and triggering antiviral, antiproliferative and immunomodulatory activity.
Interferon-β 1b, specifically, Betaseron (h-IFN-β 1b C17S), has been used to treat diseases including multiple sclerosis (MS), hepatitis B and C infections, glioma, and melanoma. Interferon-β has been demonstrated to reduce the number of attacks suffered by patients with relapsing and remitting MS. Substantial amounts of interferon-β 1b are needed for therapeutic use. Recombinant interferon-β 1b has been produced at low levels in mammalian cells, including human fibroblasts and CHO cells. Animal cell expression is typically hindered by technical difficulties including longer process time, easy contamination of cultures, a requirement for maintaining stringent culturing conditions, and the high cost of culture media. As the glycoprotein component has been found to be generally unnecessary for the activity of interferon β, research has turned to the expression of the recombinant protein in the bacterial expression system, E. coli. As noted, the inclusion bodies generated in E. coli must be solubilized by denaturation, and the interferon-β refolded. Refolding, which is slow, extends process time, adds cost, and lowers yield. To date, a method for quickly and economically producing high levels of soluble recombinant interferon-β in either mammalian or bacterial host cells, without the need for denaturing and refolding steps, has not been described.